The soluble protease ADAMDEC1 released from activated platelets hydrolyzes platelet membrane pro-EGF to active high molecular weight EGF

Platelets are the sole source of EGF in the circulation, yet how EGF is stored or released from stimulated cells are undefined. In fact, we found platelets did not store EGF, synthesized as a single 6 kDa domain in proEGF, but rather expressed intact pro-EGF precursor on granular and plasma membranes. Activated platelets released highmolecular-weight (HMW)-EGF, produced by a single cleavage between the EGF and transmembrane domains of pro-EGF. We synthesized a fluorogenic peptide encompassing residues surrounding the putative sessile arginyl residue and found stimulated platelets released a soluble activity that cleaved this pro-EGF1020-1027 peptide. High throughput screening identified chymostatins, bacterial peptides with a central cyclic arginyl structure, as inhibitors of this activity. In contrast, the matrix metalloproteinase/TACE inhibitor GM6001 was ineffective. Stimulated platelets released the soluble protease ADAMDEC1, recombinant ADAMDEC1 hydrolyzed proEGF1020-1027, and this activity was inhibited by chymostatin and not GM6001. Biotinylating platelet surface proteins showed ADAMDEC1 hydrolyzed surface pro-EGF to HMW-EGF that stimulated HeLa EGF receptor (EGFR) reporter cells, and EGFRdependent tumor cell migration. This proteolysis was inhibited by chymostatin and not GM6001. Metabolizing pro-EGF R1023 to citrulline with recombinant polypeptide arginine deiminase 4 (PAD4) abolished ADAMDEC1-catalyzed pro-EGF1020-1027 peptidolysis, while pretreating intact platelets with PAD4 suppressed ADAMDEC1-, thrombin-, or collagen-induced release of HMW-EGF. We conclude activated platelets release ADAMDEC1 that hydrolyzes proEGF to soluble HMW-EGF, HMW-EGF is active, proteolytic cleavage of pro-EGF first occurs at the carboxyl terminal arginyl residue of the EGF domain, and proteolysis is the regulated and rate-limiting step in generating soluble EGF bioactivity. ____________________________________ EGF is the prototypical growth-promoting cytokine (1,2), but also has well-established roles in stimulating tumor-initiating stem cells and tumorigenesis (3). Platelets receive immunoreactive EGF from megakaryocytes http://www.jbc.org/cgi/doi/10.1074/jbc.M116.771642 The latest version is at JBC Papers in Press. Published on April 28, 2017 as Manuscript M116.771642 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from ADAMDEC1 proteolysis of pro-EGF 2 during thrombopoiesis (4), with submaxillary glands and kidney being the major sources of EGF message (5,6). Activated platelets (7-9) are the sole source of EGF in the circulation (1013). Cytokines, growth factors, and small molecules released from activated platelets promote growth and migration of vascular cells (1,10), and EGF accounts for a portion of the salutary effects of this material on endothelial cell proliferation (14), macrophage activation in atherosclerotic plaques (15), and vessel remodeling (16). However EGF release from platelets is significantly delayed relative to degranulation (7,9,17), leaving EGF storage and release from its only source in the circulation uncharacterized. EGF is synthesized as a single 6 kDa domain within its ~140 kDa pro-EGF precursor (Fig. 1A), with the EGF domain separated from the single trans-membrane domain by a short 10 residue spacer sequence (1,2). Indeed, all EGF family members are synthesized as single domains of their pro-proteins, although there is no conserved sequence defining the amino or carboxyl proteolytic sites that flank the growth factor domains. In general, the P1 sessile residues (cleavage occurs between the P1 and P1’ residues) in these growth factor precursors tend to be small or hydrophobic (1,2,18). These types of residues are preferred by the matrix metalloproteinase family, and membrane-bound ADAM10 and ADAM17 (TACE) of the ADAM subfamily of matrix metalloproteinases proteolyze and solubilize most pro-EGF family members (19-21). However pro-EGF itself is a poor substrate for these ADAM proteases. Incompletely understood events activate membrane-bound proteases that then become competent to solubilize membrane-bound proEGF family members by cleavage between the cytokine domain and a spacer sequence that separates the EGF family member from the transmembrane domain. There is a single soluble member of the ADAM family, ADAMDEC1 (A Disintegrin And Metalloproteinase Domain-Like Protein Decysin-1) (22,23), that would overcome the stearic difficulty posed by the juxtamembrane cleavage of a membrane-bound growth factor precursor by a membrane-bound protease, but this enzyme has a mutated Znbinding site (23) and displays only dampened proteolytic activity (24). Pro-EGF differs from its family members in that the P1 residue for both the N-and Cterminal cleavage is the positively charged residue arginine (2). In fact, the first purification of EGF by Dr. Cohen in the 1960s and 1970s found both low and high molecular weight forms of EGF (25), along with a co-purifying arginyl esterase that was “postulated to function in the enzymatic liberation of EGF from a precursor” (26). The identity of this protein remains obscure, but since platelets are the source of EGF in the circulation, then platelets or their megakaryocyte precursors should contain a protease to process pro-EGF to active growth factor whether or not it is the previously purified arginyl esterase. The reduced proteome of platelets contains just four ADAM protease family members including ADAM10 and ADAM17 (27,28), suggesting that one of these four proteases hydrolyzes the sessile arginyl bonds of pro-EGF to form active cytokine. We determined how platelets store and release EGF to find that platelets do not actually store fully processed and soluble EGF, but rather expressed the pro-EGF precursor on their surface and in granules. Activated platelets released soluble ADAMDEC1 that proteolyzed surface pro-EGF at the appropriate sessile arginyl residue in the spacer sequence to generate soluble high molecular weight (HMW)EGF. HMW-EGF was an effective ligand for its EGF receptor (ErbB1, Her1), and promoted migration and invasion of untransformed headand-neck tumor cells.